Direct Cellular Effects of Blood Coagulation Proteases

这个独立之路（K99/ROO）职业发展奖申请的主要科学目标 是通过研究新的血栓形成和炎症疾病的分子病理生理学， 维生素K依赖性凝血蛋白酶的细胞保护作用机制。本申请 最初侧重于膜受体在细胞蛋白C途径调节中的作用， 随后探讨了凝固蛋白酶的细胞保护活性的新机制。主要 申请人的职业发展目标是扩大他的技术和学术经验所需的 成功转型为独立调查员这些研究将提供机会， 成功申请未来独立NIH R 01基金的基础，重点是分子机制 研究集中在凝血和炎症的交叉点上。新的假说的功能 凝血蛋白酶的细胞保护作用的蛋白质组学将使用生物化学和 细胞生物学方法。临床和治疗的影响，拟议的研究是明确的， 在大型临床试验中，活化蛋白C（ARC），而不是其他抗凝剂降低了严重 这意味着APC的抗凝活性和对脓毒症患者的直接活性的独特组合， 细胞是APC成功的基础。我发表的工作和未发表的初步数据直接导致了 这些研究为我的假设提供了有力的支持。为了验证这些假设，我提出：1） 为了表征血栓调节蛋白之间内皮细胞膜受体复合物的形成， APC产生所需的内皮蛋白C受体和蛋白酶激活受体-1和APC 对细胞的直接影响; 2）阐明血小板的潜在有益和有害的功能特性 第4因子对APC产生的影响以及APC对细胞的直接作用; 3）确定新的主题和机制 通过探索APC和fVIIa之间的相似性和差异， APC和fVIIa抗细胞凋亡活性;和4）确定甲唑凝血酶是否具有抗细胞凋亡活性。 活动，如预测的，如果这种活动需要辅因子依赖性和PAR依赖性机制。如果 建议的研究是成功的，他们将增加我们的知识，并可能导致改善治疗a 血栓形成、细胞凋亡和炎症是多种疾病的发病机制。

The major scientific goal of this Pathway to Independence (K99/ROO) Career Development Award application is to understand the molecular pathophysiology of thrombotic and inflammatory disorders by studying novel mechanisms for cytoprotective actions of vitamin K-dependent coagulation proteases. This application focuses initially on the role of membrane receptors in the regulation of the cellular protein C pathway and later on the exploration of novel mechanisms for cytoprotective activities of coagulation proteases. The major career development goal of the applicant is to expand his technical and academic experience required for a successful transition into an independent investigator. These studies will provide the opportunity and solid basis to apply successfully for future independent NIH R01 funding focused on the molecular mechanistic studies centered on the crossroads of coagulation and inflammation. Novel hypotheses on the functional proteomics of cytoprotective actions by blood coagulation proteases will be tested using biochemical and cellular biology methods. The clinical and therapeutic implications of the proposed studies are clear from the large clinical trials, where activated protein C (ARC), but not other anticoagulants reduced mortality in severe sepsis patients and implied that the unique combination of APC's anticoagulant activity and direct activity on cells is the basis for APC's success. My published work and unpublished preliminary data lead directly to the proposed studies and provide strong support for my hypotheses. In testing these hypotheses, I propose: 1) To characterize the formation of endothelial cell membrane receptor complexes between thrombomodulin, endothelial protein C receptor and protease activated receptor-1 required for APC generation and APC's direct effects on cells; 2) To clarify the potential beneficial and detrimental functional properties of platelet factor 4 for APC generation and APC's direct effects on cells; 3) To identify novel themes and mechanisms for APC and fVlla cytoprotective actions on cells by exploration of the similarities and differences between APC and fVlla anti-apoptotic activities; and 4) To establish whether meizothrombin has anti-apoptotic activity, as predicted, and if this activity requires cofactor-dependent and PAR-dependent mechanisms. If the proposed studies are successful, they will increase our knowledge and may lead to improved treatment of a variety of disorders in which thrombosis, apoptosis and inflammation contribute to pathogenesis.